JP2006317544A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope capable of improving the efficiency of utilizing light. <P>SOLUTION: The confocal microscope comprises: a means for concentrating illumination light on a point [5] of interest of a sample and points [1], [9] of non-interest positioned in the vicinity of the point of interest to simultaneously illuminating the point of interest and the points of non-interest; a means for receiving the light incident on a light receiving part confocal with the point of interest without discrimination among the light generated from the point of interest and the light generated from the points of non-interest to output a light receiving signal corresponding to intensity of the lights; a means for changing the number of the points of non-interest and taking the light receiving signals in before and after the change; and a generation means for generating a confocal signal corresponding to the intensity of light generated from the point of interest on the basis of the relationship between the plurality of light receiving signals taken in and the number of the points of non-interest changed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a confocal microscope that performs confocal observation of a sample.

In a confocal microscope, illumination light is condensed on one point of a sample, the intensity of light generated from the one point is measured by a sensor, and the same operation is repeated while two-dimensionally scanning the measurement point on the sample. Therefore, a high-resolution image intensity distribution of the sample can be obtained, and confocal observation can be performed. In order to efficiently perform confocal observation, it has been proposed to use a multipoint scanning method using, for example, a Nippon disc (see, for example, Patent Document 1). In this case, the interval between adjacent measurement points is determined according to the pinhole interval of the Nippon disk so that the light generated from each measurement point does not mix on the sensor (that is, the confocal effect is maintained). Is set.
JP 9-325279 A

However, in the above multi-point scanning method, the aperture ratio of the pinhole in the Nippon disk is low, and only a small amount of light passing through the pinhole from the light source side is guided to the sample side as illumination light. The efficiency was low.
The objective of this invention is providing the confocal microscope which can improve the utilization efficiency of light.

  The confocal microscope of the present invention condenses illumination light on each of a sample point of interest and one or more non-point of interest located in the vicinity of the point of interest, and the point of interest and the non-point of interest simultaneously. No distinction is made between illuminating means for illuminating, and light incident on the light receiving unit conjugate with the target point, among light generated from the target point and light generated from the non-target point when illuminated by the illuminating unit A light receiving means for receiving the light and outputting a light receiving signal corresponding to the intensity of the light; and the light receiving means for controlling the illumination means to change the number of the non-attention points, and outputting the light receiving from the light receiving means before and after the change. Based on the relationship between the control means for sequentially taking in signals, the plurality of received light signals taken in by the control means, and the number of changed non-attention points, a shared value corresponding to the intensity of light generated from the attention points is obtained. Generating means for generating a focus signal Than it is.

  Further, the generation means generates the confocal signal, generates a non-confocal signal according to the intensity of light generated from the non-focused point, and the confocal signal and the non-confocal signal It is preferable to generate the difference signal.

  According to the confocal microscope of the present invention, the light utilization efficiency can be improved.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
(First embodiment)
The confocal microscope 10 of 1st Embodiment is comprised by the illumination system (11-16), the imaging system (13-17), the photodetector 18, and the control part 19 as shown in FIG. . The illumination system (11 to 16) includes a light source 11, a condenser lens 12, a dichroic mirror 13, a mirror element 14, and lenses 15 and 16. The imaging system (13 to 17) includes the lenses 15 and 16, the mirror element 14, the dichroic mirror 13, and the lens 17.

  The mirror element 14 includes a large number of two-dimensionally arranged micromirrors 4A (see also FIG. 2), and each tilt angle can be adjusted at high speed (for example, DMD; Digital Micromirror Device). The photodetector 18 is an image pickup device such as a CCD, for example, and has a large number of light receiving portions 8A arranged two-dimensionally (FIG. 2). Each light receiving unit 8A is composed of, for example, one or more pixels of an image sensor. The mirror element 14 and the photodetector 18 are arranged in a conjugate plane with each other, and are also arranged in a conjugate manner with the sample surface 20.

  In FIG. 2, each light receiving portion 8 </ b> A of the photodetector 18 is shown corresponding to each micromirror 4 </ b> A of the mirror element 14. Furthermore, the sample surface 20 is shown divided into a large number of minute regions 2A corresponding to the two-dimensional array of minute mirrors 4A (two-dimensional array of light receiving portions 8A). Each micro region 2A is conjugate with each micro mirror 4A, and is also conjugate with each light receiving portion 8A. In FIG. 2, the micro area 2 </ b> A, the micro mirror 4 </ b> A, and the light receiving unit 8 </ b> A are shown to have the same size, but the actual sizes do not always match due to the focal lengths of the lenses 15 to 17. Absent.

  The light from the light source 11 passes through the condenser lens 12 and the dichroic mirror 13 and then uniformly illuminates the numerous micromirrors 4A of the mirror element 14. Some of the small number of micromirrors 4A are set to illumination tilt angles (hereinafter referred to as “on-state micromirrors 4A”), and the rest are set to non-illumination tilt angles different from the illumination tilt angles. (Hereinafter, “micromirror 4A in the off state”). Switching on / off of the micromirror 4A is performed at high speed based on a control signal from the control unit 19.

  Among the many micromirrors 4A, the micromirror 4A in the on state reflects light from the dichroic mirror 13 and guides it toward the lens 15. The light passes through the lenses 15 and 16 and then enters the sample surface 20 as illumination light. The incident position of the illumination light on the sample surface 20 is a minute area 2A conjugate with the on-state minute mirror 4A in the mirror element 14 among the many minute areas 2A. The illumination light incident on the minute area 2A is collected at one point. The light reflected by the micro mirror 4A in the off state does not reach the sample surface 20.

  In the minute area 2A where the illumination light is incident on the sample surface 20, the fluorescent material is excited by the illumination light, and fluorescence is generated from the fluorescent material. After passing through the lenses 16 and 15, the fluorescence is incident on the minute mirror 4 A in the on state of the mirror element 14, reflected there, and guided to the dichroic mirror 13, and is detected through the dichroic mirror 13 and the lens 17. Incident on the vessel 18. The incident position of the fluorescence in the photodetector 18 is the light receiving portion 8A conjugate with the micromirror 4A in the ON state of the mirror element 14 among the many light receiving portions 8A.

  As described above, in the confocal microscope 10 according to the first embodiment, among the many micro regions 2A on the sample surface 20, the micro region 2A conjugate with the micro mirror 4A in the on state of the mirror element 14 is illuminated, and the micro region 2A is illuminated. The fluorescence generated from the region 2A is reflected by the on-state micromirror 4A, guided to the photodetector 18, and detected by the light receiving unit 8A conjugate with the on-state micromirror 4A. For this reason, fluorescence by an arbitrary illumination pattern can be detected by switching on / off the micromirror 4A.

  For example, when the illumination pattern shown in FIG. 3A is formed on the sample surface 20, the sample surface 20 is illuminated by each of a plurality of minute regions 2A separated by a certain distance D1. In FIG. 3A, the white area corresponds to the minute area 2A illuminated, and the hatched area corresponds to the minute area 2A not illuminated. Then, the fluorescence from the illuminated minute region 2A (hereinafter “attention point 2A”) is detected by the light receiving unit 8A conjugate to each attention point 2A. At this time, if the distance D1 between the adjacent attention points 2A is set so that the confocal effect is maintained, and the attention point 2A is two-dimensionally scanned on the sample surface 20, a high-resolution image intensity distribution on the sample surface 20 can be obtained. Can be obtained and confocal observation is possible.

  In the following description, the interval D1 in which the confocal effect is maintained is, for example, twice the interval D2 between the minute regions 2A. Further, each of the plurality of attention points 2A separated by the distance D1 (= D2 × 2) and the eight minute regions 2A adjacent thereto are collectively referred to as a unit 21 (FIG. 3B). Each unit 21 includes a total of nine minute regions 2A in a 3 × 3 array, and the center thereof is the attention point 2A. In FIG. 3B, the numbers “1” to “9” are assigned to the nine minute regions 2A in the unit 21 for the sake of convenience.

  When only the “5” attention point 2A in the unit 21 is illuminated as in the illumination pattern of FIG. 3B, the fluorescence generated from the adjacent attention points 2A is adjacent to each attention point 2A. The light enters the matching light receiving portions 8A and does not mix with each other. For this reason, the signal output from one light-receiving part 8A becomes a confocal signal corresponding to the intensity of the fluorescence generated from one attention point 2A. Further, the signal output from the other light receiving unit 8A is a confocal signal corresponding to the intensity of the fluorescence generated from the other target point 2A.

  In contrast to this, for example, as shown in the illumination pattern of FIG. 4A, not only the “5” attention point 2A in the unit 21, but also the minute areas 2A of “1” and “9” in the vicinity thereof (hereinafter “non-attention”). The situation is different if the point 2A ") is also illuminated at the same time. The interval between the attention point 2A and the non-attention point 2A in the unit 21 is a narrow interval where the confocal effect is not maintained, and the light receiving unit 8A conjugate to the attention point 2A has not only the fluorescence generated from the attention point 2A. A part (leakage light) of each fluorescence generated from the non-attention point 2A also enters.

  The light receiving unit 8A conjugate to the point of interest 2A receives the fluorescence from the point of interest 2A and the leaked light from the non-point of interest 2A without distinguishing them, and outputs a light reception signal corresponding to the intensity of the light. The light reception signal in this case is the sum of the confocal signal according to the intensity of the fluorescence from the point of interest 2A and the non-confocal signal according to the intensity of the leaked light from the non-point of interest 2A. Therefore, it is not possible to obtain a high-resolution image intensity distribution on the sample surface 20 using the received light signal itself.

  However, for example, by receiving the same light reception signal using the illumination pattern of FIG. 4B and performing predetermined signal processing in combination with the light reception signal acquired using the illumination pattern of FIG. The non-confocal signal can be subtracted from to extract only the confocal signal. For this reason, by using the confocal signal extracted by the signal processing, a high-resolution image intensity distribution of the sample surface 20 can be obtained.

  In the signal processing, the magnitude of the non-confocal signal included in the light reception signal is assumed as follows. That is, as in the illumination pattern of FIG. 5, all the minute regions 2A other than the attention point 2A of “5” in the unit 21 (non- attention points 2A of “1” to “4”, “6” to “9”). Is assumed to be simultaneously illuminated, and the magnitude of the received light signal output from the light receiving unit 8A conjugate to the point of interest 2A is “b”. This light reception signal (magnitude b) is a non-confocal signal corresponding to the total intensity of leaked light from the non-target point 2A of “1” to “4” “6” to “9” in the unit 21. It is considered that light leaked from outside the unit 21 is not mixed.

  Furthermore, in order to simplify the description, it is assumed that the contribution from each non-attention point 2A to the received light signal is equal. In this case, the magnitudes of the non-confocal signals caused by the non-focused points 2A of “1” to “4”, “6” to “9” are equal to each other, and average “(1/8) b” is obtained. Become. Further, the magnitude of the received light signal when the “5” attention point 2A in the unit 21 is illuminated as in the illumination pattern of FIG. This received light signal (magnitude a) is equal to a confocal signal corresponding to the intensity of fluorescence from the point of interest 2A.

When using the magnitude a of the confocal signal due to the attention point 2A in the unit 21 and the magnitude (1/8) b of the non-confocal signal due to each non-attention point 2A, FIG. ), The magnitude S1 of the received light signal when the attention point 2A of “5” and the two non-interesting points 2A of “1” and “9” are simultaneously illuminated is expressed by the following equation (1) ).
S1 = a + (1/8) b × 2 (1)
Next, the case of the illumination pattern in FIG. 4B will be described. In the illumination pattern of FIG. 4B, the attention point 2A of “5” in the unit 21 and the four non-interesting points 2A of “2”, “4”, “6”, and “8” in the vicinity thereof are simultaneously illuminated. is doing. In this case, the magnitude S2 of the received light signal output from the light receiving unit 8A conjugate to the point of interest 2A can be expressed by the following equation (2).

S2 = a + (1/8) b × 4 (2)
In the confocal microscope 10 of the present embodiment, for example, when the control unit 19 controls the mirror element 14 to form the illumination pattern of FIG. The light receiving signal (magnitude S1) output from the light receiving unit 8A conjugate with the point of interest 2A is captured, and then the number of non-attention points 2A is changed (2 → 4), and the illumination shown in FIG. When the pattern is formed on the sample surface 20, a light reception signal (magnitude S2) output from the light receiving unit 8A conjugate with the target point 2A of “5” in the unit 21 is captured.

  In this way, when the received light signals (magnitudes S1 and S2) when the illumination states other than the point of interest 2A in the unit 21 (the number of non-points of interest 2A) are different are sequentially taken in, the control unit 19 The above equation (1) representing the relationship between the received light signal (magnitude S1) and the number (two) of the non-target points 2A, the other received light signal (magnitude S2) and the number of the non-target points 2A ( 4), the confocal signal (magnitude a) corresponding to the intensity of the fluorescence from the point of interest 2A is generated.

  The confocal signal (magnitude a) generated using the two types of illumination patterns shown in FIGS. 4A and 4B is a signal related to the attention point 2A of “5” in the unit 21, and FIG. It corresponds to the light reception signal by the illumination pattern of (b). Similarly, another small area 2A of “9”, for example, is set as the point of interest 2A, and the unit 22 centered on this is considered, and two types of illumination patterns shown in FIGS. 6 (a) and 6 (b) are used. The confocal signal (magnitude a) related to the attention point 2A is generated.

  Similarly, the remaining small regions 2A of “1” to “4”, “6” to “8” are similarly set as attention points 2A in order, and two types of illumination patterns (the number of non-attention points 2A is different) are used. , A confocal signal (magnitude a) related to each attention point 2A is generated. In this way, by performing two-dimensional scanning of the point of interest 2A on the sample surface 20, a high-resolution image intensity distribution on the sample surface 20 can be obtained, and confocal observation is possible.

  Further, in the confocal microscope 10 of the present embodiment, a plurality of attention points 2A arranged at the interval D1 (= D2 × 2) where the confocal effect is maintained on the sample surface 20 is illuminated, and each attention point 2A is illuminated. In comparison with the confocal microscope 10 using the conventional nippo disk, the light from the light source 11 is used to simultaneously illuminate the non-focused points 2A arranged at a narrow interval where the confocal effect is not maintained. Efficiency is improved. As a result, the S / N ratio is improved.

Furthermore, in the confocal microscope 10 of the present embodiment, there are a plurality of optical paths that guide the illumination light from the light source 11 toward the sample surface 20 and optical paths that guide the fluorescence from the sample surface 20 toward the photodetector 18. Since the mirror element 14 (FIG. 2) having the micromirror 4A is also used, the apparatus can be simplified.
The procedure for two-dimensionally scanning the target point 2A on the sample surface 20 by switching on / off the mirror element 14 can be simplified as follows. The illumination pattern of the unit 22 in which the “9” minute region 2A of the sample surface 20 shown in FIG. 6A is the point of interest 2A and the “5” “1” minute region 2A is the non-target point 2A is “5”. If it replaces with the unit 21 centering on 2A of this area, it will become as shown in FIG.6 (c) and it turns out that it is the same as the illumination pattern of the unit 21 shown to Fig.4 (a).

  For this reason, when the illumination pattern of the unit 21 shown in FIG. 4A is formed on the sample surface 20, the light reception signals (size S1) from the respective light receiving portions 8A conjugate with “5” and “9” in the unit 21. ), The formation of the illumination pattern of the unit 22 shown in FIG. 6A can be omitted. In addition, when the illumination pattern of FIG. 4A is formed, it is preferable to capture a light reception signal (size S1) also from the light receiving portion 8A conjugate with “1” in the unit 21.

  When the illumination pattern of FIG. 4 (a) is formed, the light reception signal (size S1) from each light receiving portion 8A conjugate to the illuminated minute region 2A (“1” “5” “9”) in the unit 21 is formed. ) And the confocal signal (magnitude a) is generated with each minute region 2A as the point of interest 2A, the received light signal (magnitude S1) may be used. For example, the received light signal (magnitude S1) from the light receiving unit 8A conjugate to the minute area 2A of “1” is used when a confocal signal (magnitude a) is generated with “1” as the attention point 2A.

  Similarly, when illuminating the minute areas 2A of “2”, “6”, and “7” in the unit 21 as in the illumination pattern of FIG. 7A, each light receiving unit conjugate to these minute areas 2A. When the received light signal (magnitude S1) is taken from 8A and the confocal signal (magnitude a) is generated with “2”, “6”, and “7” as the attention point 2A, the received light signal (magnitude S1) is used. That's fine. In the case of the illumination pattern of FIG. 7B, when the confocal signal (magnitude a) is generated with “3”, “4”, and “8” as the attention point 2A, the received light signal (magnitude S1) is used. That's fine.

As described above, when the two minute regions 2A at the upper left and lower right in the figure are set as the non-attention points 2A with respect to the attention point 2A, FIG. 4 (a), FIG. 7 (a), (b ), The light receiving signals (magnitude S1) relating to all the minute regions 2A (“1” to “9”) in the unit 21 are efficiently captured simply by forming the three illumination patterns shown in FIG. be able to.
However, the unit 22 in which the minute region 2A of “9” on the sample surface 20 shown in FIG. 6B is set as the attention point 2A and the minute region 2A of “3”, “6”, “7”, and “8” is set as the non-target point 2A. When the illumination pattern is replaced with the unit 21 centered on the minute region 2A of “5”, the illumination pattern is as shown in FIG. 6D, which is different from the illumination pattern of the unit 21 shown in FIG. . For this reason, when the four minute regions 2A located on the top, bottom, left, and right of the attention point 2A are set as the non-attention points 2A, the nine illumination patterns shown in FIGS. 4B, 6B, and 8 are used. It is necessary to form the sample surface 20 in order.

In the first embodiment described above, the case where the contributions (that is, the magnitude of the non-confocal signal) to the received light signal from each of the eight non-target points 2A located in the vicinity of the target point 2A is described as an example. However, the present invention is not limited to this. The present invention can also be applied when weighting is performed according to the positional relationship between the attention point 2A and the non-attention point 2A, and the magnitude of the non-confocal signal is varied.
For example, when “5” is the attention point 2A and “1” to “4”, “6” to “9” are the non-attention points 2A, the non-confocal signal caused by the non-attention point 2A of the number “N” Is set to be “K _N × b” and the sum of the coefficients K _N (= ΣK _N ) becomes “1”.
(Second Embodiment)
Here, a plurality of illumination patterns (FIGS. 9 and 10) having the same number of non-target points 2A and different arrangements will be described.

  In each of the four illumination patterns shown in FIG. 9, the “5” minute region 2A in the unit 21 is the point of interest 2A, and the two minute regions 2A in the vicinity thereof are the non-notable points 2A. Although the arrangement of the non-attention points 2A is different for each illumination pattern, by forming these four illumination patterns in order on the sample surface 20, eight minute regions 2A ("1" to "1" to the vicinity of the attention point 2A) are formed. "4", "6" to "9") can be illuminated one by one in order.

In this case, the light receiving signals (magnitudes S1 (1) to S1 (4)) output from the light receiving unit 8A conjugate with the target point 2A of “5” are sequentially taken in by each illumination pattern of FIG. Can be expressed by the following equation (3).
S3 = S1 (1) + S1 (2) + S1 (3) + S1 (4)
= A * 4 + (1/8) b * 8 = 4a + b (3)
In each of the six illumination patterns shown in FIG. 10, the “5” minute region 2A in the unit 21 is the attention point 2A, and the four minute regions 2A in the vicinity thereof are the non-notice points 2A. Although the arrangement of the non-attention points 2A is different for each illumination pattern, by forming these six illumination patterns on the sample surface 20 in order, eight minute regions 2A (“1” to “1” in the vicinity of the attention point 2A). 4 ”,“ 6 ”to“ 9 ”) can be illuminated three times in order.

In this case, the received light signals (magnitudes S2 (1) to S2 (6)) output from the light receiving unit 8A conjugate with the point of interest 2A of “5” are sequentially taken in by each illumination pattern of FIG. Can be expressed by the following equation (4).
S4 = S2 (1) + S2 (2) + S2 (3) + S2 (4) + S2 (5) + S2 (6)
= A * 6 + (1/8) b * 24 = 6a + 3b (4)
In the second embodiment, when the received light signals (magnitudes S3 and S4) when the illumination states other than the point of interest 2A in the unit 21 (the number of non-points of interest 2A) are different are sequentially obtained, (S3) and the number of non-target points 2A (2), the above equation (3), the other received light signal (size S4) and the number of non-target points 2A (4) The above equation (4) representing the above relationship is combined to generate a confocal signal (magnitude a) corresponding to the intensity of fluorescence from the point of interest 2A.

  As can be seen from the above formulas (3) and (4), the terms of the non-confocal signal are all the minute regions 2A other than the attention point 2A of “5” (“1” to “4” in FIG. 5). It is an integral multiple of the non-confocal signal (magnitude b) corresponding to the total intensity of leakage light from the non-focused points 2A) of “6” to “9”. For this reason, even when the contribution to the received light signal differs depending on the positional relationship between the attention point 2A and the non-attention point 2A, the confocal signal (magnitude a) relating to the attention point 2A is not affected by the difference. ) Can be generated with good reproducibility.

  The confocal signal (magnitude a) generated using the ten types of illumination patterns shown in FIGS. 9 and 10 is a signal related to the attention point 2A of “5” in the unit 21. Similarly, the remaining small regions 2A of “1” to “4”, “6” to “9” are similarly set as attention points 2A in order, and 10 types of illumination patterns (the number of non-attention points 2A is different) are used. A confocal signal (magnitude a) relating to each attention point 2A may be generated.

However, even in this case, there are illumination patterns that can be shared, as described with reference to the comparison of the illumination patterns in FIGS. 4A and 5A. Therefore, in addition to the 10 types of illumination patterns shown in FIGS. 9 and 10, if the 13 types of illumination patterns shown in FIGS. 11 and 12 are sequentially formed, all necessary illumination patterns are covered.
The received light signals (magnitude S3) related to the minute regions 2A of “1” to “9” in the unit 21 can be efficiently obtained using the 12 illumination patterns shown in FIGS. In addition, the received light signals (magnitude S4) relating to the minute area 2A of “1” to “9” can be efficiently obtained using the 11 illumination patterns shown in FIGS.

As described above, in the second embodiment, the point of interest 2A is two-dimensionally scanned on the sample surface 20 using a plurality of illumination patterns (FIG. 9, FIG. 10, etc.) having the same number of non-target points 2A and different arrangements. A high-resolution image intensity distribution on the sample surface 20 can be obtained with good reproducibility.
Also in the second embodiment, a plurality of attention points 2A arranged at a distance D1 (= D2 × 2) at which the confocal effect is maintained on the sample surface 20, and the confocal effect for each attention point 2A. Since the non-attention points 2A arranged at a narrow interval that cannot be maintained are simultaneously illuminated, the light use efficiency from the light source 11 is improved.

The light use efficiency E in this case is as shown in the following equation (5) when an average value is obtained in consideration of the time taken to sequentially form all the illumination patterns (23 types).
E = {(12/23) ・ [3/9] + (1/23) ・ [4/9] + (10/23) ・ [5/9]} × [10/23]
= 0.189 (5)
Numeric molecules (3,4,5) in parentheses [] are the number of illuminated microregions 2A, and numeric molecules (12,1,10) in parentheses () are the number of illuminated microregions 2A. The numerator (10) in parentheses [] represents the number of illumination patterns used in the calculation to generate a confocal signal.

In order to compare with the light utilization efficiency shown in Equation (5), when only a plurality of attention points 2A at a distance D1 (= D2 × 2) where the confocal effect is maintained is illuminated as in the conventional case (FIG. 13). The utilization efficiency E ′ was determined in the case of using nine types of illumination patterns shown. The result is as shown in the following equation (6).
E ′ = {(9/9) · [1/9]} × [1/9]
= 0.0123 (6)
As can be seen from the above numerical calculation, the light use efficiency from the light source 11 is remarkably improved in the second embodiment. As a result, the S / N ratio is improved and good confocal observation is possible.
(Third embodiment)
When the control unit 19 generates the confocal signal (magnitude a) by the simultaneous equations (1) and (2) (or the simultaneous equations (3) and (4)), the non-confocal signal (magnitude) B) is also generated. The non-confocal signal (magnitude b) is obtained from all the minute regions 2A other than the attention point 2A (for example, eight non- attention points 2A of “1” to “4”, “6” to “9” shown in FIG. 5). It is a signal according to the total intensity of the leaked light from.

  Further, a difference signal (ab) between the confocal signal (magnitude a) and the non-confocal signal (magnitude b) is generated to obtain an image intensity distribution on the sample surface 20. The difference signal (ab) is considered to represent information of a region narrower than the confocal signal (magnitude a) on the sample surface 20. For this reason, the image intensity distribution on the sample surface 20 corresponding to the difference signal (ab) has higher resolution than the image intensity distribution corresponding to the confocal signal (magnitude a). This principle will be described qualitatively.

For example, when only the point of interest 2A of “5” is illuminated and the point of interest 2A is located on the optical axis as in the illumination pattern of FIG. 14 (a) (see FIGS. 14 (b) and 14 (c)), the point of interest The light receiving signal output from the light receiving unit 8A conjugate with 2A is a confocal signal (size a). The illumination area inside the sample surface 20 has a shape as shown in FIG.
On the other hand, as shown in the illumination pattern of FIG. 14D, “2” and “8” non-target points 2A located in the vicinity of the “5” target point 2A are simultaneously illuminated, and the target point 2A is positioned on the optical axis. (See FIGS. 14E and 14F), the received light signal output from the light receiving unit 8A conjugate with the point of interest 2A is a non-confocal signal (size b). Further, the illumination area inside the sample surface 20 has a shape as shown in FIG.

Information on the sample surface 20 included in the difference signal (ab) between the confocal signal (magnitude a) and the non-confocal signal (magnitude b) is shown in FIGS. 14 (g) and 14 (h). It can be seen that the information corresponds to a hatched portion and is narrower than the information in FIGS. 14 (b) and 14 (c). Therefore, the resolution in the optical axis direction and in the plane can be improved by performing the above difference processing.
In order to quantitatively show the effect of the difference processing, the optical transfer function of the image intensity distribution corresponding to the difference signal (ab) is shown in FIG. For comparison, the optical transfer function of the image intensity distribution according to the confocal signal (magnitude a) is shown in FIG. The horizontal axis in FIG. 15 represents the frequency Nz [1 / μm] in the optical axis direction. The vertical axis is the optical transfer function (OTF).

In this simulation, difference processing was performed using the non-confocal signals in FIGS. The wavelength was 0.55 μm, the numerical aperture was 0.75, and the magnification was 40 times. The size of the minute region 2A was set to 25 μm so that light leaked from the outside of the unit 21 can be almost ignored. As can be seen from the comparison between FIGS. 15 (a) and 15 (b), the optical transfer function (a) when the difference processing is performed in the frequency Nz range of 0.0 to 0.8 has a larger value. In comparison with the case (b) in which the difference processing is not performed, the optical characteristics are better.
(Modification)
In the above-described embodiment, the example using the illumination pattern including the two non-target points 2A and the illumination pattern including the four non-target points 2A has been described, but the present invention is not limited to this. The present invention can be applied when the number of non-attention points 2A is one or more and two or more types of illumination patterns having different numbers of non-attention points 2A are used. Further, in one of a plurality of illumination patterns having different numbers of non-attention points 2A, the illumination on the non-attention points 2A may be blocked. Furthermore, when using three or more types of illumination patterns with different numbers of non-target points 2A, it is preferable to solve simultaneous equations by the method of least squares. In this case, since the amount of data is large, the accuracy is improved.

  Further, in the above-described embodiment, the example in which each unit 21 includes a total of nine minute regions 2A in a 3 × 3 array has been described, but the present invention is not limited to this. The sizes of the unit 21 and the minute area 2A are preferably determined appropriately from the size of the illumination area that affects the light reception signal. The arrangement of the units 21 may be an even number × even number, but an odd number × odd number (for example, 5 × 5) is preferable because the influence of leakage light from the non-target point 2A can be considered isotropically.

  Further, in the above-described embodiment, the example in which the reflective spatial light modulation element (for example, the mirror element 14) is used as the illumination optical path and the light reception optical path has been described, but the present invention is not limited to this. When confocal observation of the light-transmitting sample surface 20 is performed, a similar spatial light modulation element may be disposed in each of the illumination optical path and the light receiving optical path for synchronous control. Instead of the reflective spatial light modulator, a transmissive spatial light modulator (for example, a transmissive optical element having a number of two-dimensionally arranged liquid crystal cells) may be used.

1 is a diagram illustrating an overall configuration of a confocal microscope 10. It is a schematic diagram explaining the corresponding relationship among the minute mirror 4A, the light receiving unit 8A, and the minute region 2A. It is a figure explaining the illumination pattern in the case of illuminating only the attention point 2A. An illumination pattern (a) in the case of simultaneously illuminating the attention point 2A and the two non-attention points 2A, and an illumination pattern (b) in the case of simultaneously illuminating the attention point 2A and the four non-attention points 2A will be described. FIG. It is a figure explaining the illumination pattern in the case of illuminating eight non-attention points 2A. It is a figure explaining the two-dimensional scanning of the attention point 2A. It is a figure explaining the other illumination pattern in the case of illuminating the attention point 2A and the two non-notice points 2A simultaneously. It is a figure explaining the other illumination pattern in the case of illuminating the attention point 2A and the four non-notice points 2A simultaneously. It is a figure explaining the some illumination pattern from which arrangement | positioning differs in the number (two) of the non-attention points 2A used in 2nd Embodiment. It is a figure explaining the some illumination pattern from which arrangement | positioning differs in the number (four) of the non-attention points 2A used in 2nd Embodiment. It is a figure explaining the other illumination pattern in the case of illuminating the attention point 2A and the two non-notice points 2A simultaneously. It is a figure explaining the other illumination pattern in the case of illuminating the attention point 2A and the four non-notice points 2A simultaneously. It is a figure explaining the illumination pattern of a comparative example. It is a figure explaining the difference process of 3rd Embodiment. It is a figure explaining the effect of difference processing.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Confocal microscope 11 Light source 12 Condenser lens 13 Dichroic mirror 14 Mirror element 4A Micro mirror 15, 16, 17 Lens 18 Photo detector 8A Light receiving part 19 Control part 20 Sample surface 2A Micro area (attention point, non-attention point)

Claims (2)

Illuminating means for condensing illumination light on each of the sample point of interest and one or more non-point of interest located in the vicinity of the point of interest, and illuminating the point of interest and the non-point of interest simultaneously;
Of the light generated from the target point and the light generated from the non-target point when illuminated by the illuminating means, the light incident on the light receiving unit conjugate with the target point is received without distinction, and the light A light receiving means for outputting a light reception signal corresponding to the intensity of
Control means for controlling the illumination means to change the number of non-attention points, and sequentially taking in the received light signals output from the light receiving means before and after the change,
Generating means for generating a confocal signal according to the intensity of light generated from the attention point based on the relationship between the plurality of light reception signals captured by the control means and the number of changed non-attention points; A confocal microscope characterized by comprising: The confocal microscope according to claim 1,
The generation means generates the confocal signal, generates a non-confocal signal according to the intensity of light generated from the non-focused point, and a difference between the confocal signal and the non-confocal signal A confocal microscope characterized by generating a signal.

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